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| Status |
Public on Feb 02, 2016 |
| Title |
Control-2 |
| Sample type |
SRA |
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| Source name |
TK6_control
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| Organism |
Homo sapiens |
| Characteristics |
cell line: TK6
cell type: human lymphoblastoid cells
genotype/variation: heterozygous at the thymidine kinase (TK) locus
exposed to: static (control)
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| Treatment protocol |
For ground-based simulation of microgravity, HARV rotating suspension culture bioreactors (Synthecon, Houston, TX) were used. Actively growing (96–98% viable) TK6 cells were seeded in the bioreactor at 2 X 10^5 cells/ml and rotated at 12 rpm/min. For control, cells (at the same cellular density i.e 2 X 10^5 cells/ml) were maintained in bioreactors in a normal gravity (static) condition as controls
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| Growth protocol |
TK6 human lymphoblastoid cells (ATCC, Manassas, VA) were maintained in the log phase of cell growth by culturing in RPMI-1640 (Life Technologies, Grand Island, NY) medium supplemented with 10% Fetal Bovine Serum (Atlanta Biologicals, Flowery Branch, GA) and 1% Penicillin/Streptomycin (Life Technologies, Grand Island, NY) at 37 °C in 5% CO2 and 95% air
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from duplicates of cells subjected to stimulated microgravity and static control using RNA-STAT-60 (Tel-Test,Inc., Friendswood, TX) using the manufacturer’s instructions. Briefly, 1ml of RNA-STAT solution was added per 10^6 cells and homogenized for 5 minutes over ice. 1ml of chloroform was added, contents shaked vigorously and centrifuged at 12,000g at 4ºC for 15 minutes. The aqueous solution was transferred to a clean corex tube and 0.8 ml isopropanol added. After incubation of 10 minutes, the contents were centrifuged at 12,000g for another 10 minutes to precipitate the RNA. The RNA pellet was washed with 75% ethanol and centriguged ar 7500g for 5 minutes at 4ºC. The ethanol was aspirated and the RNA pellet dried. The RNA pellet was finally resuspended in DEPC water.
RNA was converted into cDNA and was end-repaired using repair solution (combination of T4 DNA polymerase, E. coli DNA Pol I large fragment and T4 polynucleotide kinase). A-tailing was obtained by incubating the end repaired DNA with dA-tailing mix. Blunt end ligation was performed by incubating the A-tailed DNA samples (1 μg) with adapters (IDT Inc., Coralville, IA), unmethylated version of Truseq adapters (Illumina Inc., San Diego, CA) for multiplexing. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the illumina hiseq2000 following the manufacturer's protocols.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
005078_D-tk6-static
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| Data processing |
Sequence reads were aligned with tophat-2.0.8.
Aligned reads were fitered with NGSUtils (Breese and Liu, Bioinformatics 2013), only unique mapped reads with no more than 2 mismatchs were kept.
Read counts were calculated with NGSUtils.
Differential expression analysis was performed using edgeR.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text file includes row counts and counts per million reads from edgeR.
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| Submission date |
Feb 13, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Joseph Irudayaraj |
| E-mail(s) |
josephi@purdue.edu
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| Phone |
765-494-0388
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| Organization name |
Purdue University
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| Department |
Bindley Bioscience Center
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| Street address |
225 S. University Street
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| City |
West Lafayette |
| State/province |
IN |
| ZIP/Postal code |
47907 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE65943 |
A study of alterations in DNA epigenetic modifications (5mC and 5hmC) and gene expression influenced by simulated microgravity in human lymphoblastoid cells [RNA-seq] |
| GSE65944 |
A study of alterations in DNA epigenetic modifications (5mC and 5hmC) and gene expression influenced by simulated microgravity in human lymphoblastoid cells |
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| Relations |
| BioSample |
SAMN03344816 |
| SRA |
SRX878654 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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